The goal of this project is to develop a new type of nonlinear-optical microscope, and to establish its potential for thick-tissue imaging. The microscope uses a femtosecond-pulsed infrared laser beam to trans-illuminate the tissue sample. As opposed to other nonlinear-optical techniques such as two-photon excited fluorescence (TPEF) microscopy, or second-harmonic generation (SHG) microscopy, the laser beam does not generate signal within the sample. Instead, the laser beam traverses the sample completely and is then focused onto a nonlinear crystal. The detected signal is the SHG produced by the crystal. The contrast mechanism is as follows: the laser beam incurs spatially dependent phase fluctuations as it traverses the sample. These phase fluctuations lead to a defocusing of the beam on the crystal, which is monitored as a reduction in the SHG signal. Because the SHG is sensitive to defocusing, the crystal acts as a virtual self-aligned confocal pinhole, thereby leading to phase sensitivity and intrinsic 3d resolution. This technique, called auto-confocal microscopy (ACM), requires no labeling, is technically simple, and may be readily combined with existing nonlinear imaging modalities such as TPEF microscopy. The R21 Phase (1 yr) will consist in building an ACM, and quantifying its performance in terms of resolution, depth-penetration, etc., using simple samples such as latex beads (or cultured cells) in agarose (or collagen) gels. The ACM is expected to reveal local index-of-refraction fluctuations throughout a sample with high temporal resolution (microsecs), and as yet uncharacterized spatial resolution depending on the sample thickness. The R33 Phase (3 yrs) will involve vertebrate-animal tissue imaging. For thin enough samples (to be determined), an ACM should rapidly assess sample structure from local (micron) to global (millimeter) length scales. Our proposed project will apply such wide dynamic-range measurements to a variety of topics including brain-slice imaging, diseased tissue screening, and the monitoring of epithelial tissue dynamics, using simultaneous ACM and TPEF contrasts.